Development of an in vitro visual assay facilitated the study of large numbers of megakaryocytes undergoing proplatelet formation in short-term cultures. Approximately 9% of megakaryocytes formed platelets during a 24-hour period. In the presence of an inhibitor of anaerobic glycolysis (NaF), proplatelet formation was inhibited, whereas inhibitors of respiration (NaCN) did not significantly (P greater than 0.05) decrease proplatelet formation. Presence of the microtubule-disrupting agents colchicine and vincristine sulfate in culture medium inhibited proplatelet formation, whereas the microfilament-disrupting agent cytochalasin B had a less pronounced inhibition.